Decreased GSH content has been found in various aged tissues and in age- related diseases, and has been suggested to be responsible for the increased oxidative damage observed in aged tissues and age-related neurodegenerative diseases. Both direct and indirect evidence indicates that decreased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, is at least one of the factors that contribute to decreased GSH in aged tissues. However, the mechanism underlying such decreased GCS activity is not clear. Therefore, the specific aims for this project are: 1) To confirm that both GCS activity and GSH content decrease in aged brain by comparing different sections of brains from 3, 12, and 24 month-old mice. 2) To determine whether the decreased GCS activity in aged tissue is due to decreased protein amount by Western blotting. 3) To explore the potential mechanism responsible for down-regulation of GCS expression in aged brain. The translation and the stability of GCS proteins from brain sections of young and aged mice will be measured using short-term [35S] methionine labeling technique. GCS mRNA contents and the transcription rates of GCS genes in young and aged brains will be measured by Northern blotting and nuclear run-on analyses, respectively. Stability of GCS mRNAs will be determined by injecting young and aged mice with transcription inhibitor actinomycin D and then measuring half- life of GCS mRNAs from the brain sections by Northern blotting analysis. Furthermore, brain slices from aged and young animals will be treated with insulin of glucocorticoid to see whether the down-regulation of GCS expression in aged brain is due to a decreased response of the cells to hormones. If no difference in the amount of GCS protein is found between brain sections from young and aged animals, structural modification of GCS protein will be examined as described in aim 4. 4) To determine whether phosphorylation and/or oxidation of GCS proteins is responsible for the decreased GCS activity in aged tissues. The long-term objective of this project is to uncover the mechanisms underlying the aging process and age-related diseases and therefore, to provide clues for potential therapeutic approaches to the age-related diseases.